Altered inflammasome activation in neonatal encephalopathy persists in childhood.

Neonatal encephalopathy (NE) is characterized by altered neurological function in term infants and inflammation plays an important pathophysiological role. Inflammatory cytokines interleukin (IL)-1ß, IL-1ra and IL-18 are activated by the nucleotide-binding and oligomerization domain (NOD)-, leucine-rich repeat domain (LRR)- and NOD-like receptor protein 3 (NLRP3) inflammasome; furthermore, we aimed to examine the role of the inflammasome multiprotein complex involved in proinflammatory responses from the newborn period to childhood in NE. Cytokine concentrations were measured by multiplex enzyme-linked immunosorbent assay (ELISA) in neonates and children with NE in the absence or presence of lipopolysaccharide (LPS) endotoxin. We then investigated expression of the NLRP3 inflammasome genes, NLRP3, IL-1ß and ASC by polymerase chain reaction (PCR). Serum samples from 40 NE patients at days 1 and 3 of the first week of life and in 37 patients at age 4-7 years were analysed. An increase in serum IL-1ra and IL-18 in neonates with NE on days 1 and 3 was observed compared to neonatal controls. IL-1ra in NE was decreased to normal levels at school age, whereas serum IL-18 in NE was even higher at school age compared to school age controls and NE in the first week of life. Percentage of LPS response was higher in newborns compared to school-age NE. NLRP3 and IL-1ß gene expression were up-regulated in the presence of LPS in NE neonates and NLRP3 gene expression remained up-regulated at school age in NE patients compared to controls. Increased inflammasome activation in the first day of life in NE persists in childhood, and may increase the window for therapeutic intervention.

Physical after-effects of colposcopy and related procedures, and their inter-relationship with psychological distress: a longitudinal survey.

OBJECTIVE: To estimate prevalence of post-colposcopy physical after-effects and investigate associations between these and subsequent psychological distress. DESIGN: Longitudinal survey. SETTING: Two hospital-based colposcopy clinics. POPULATION: Women with abnormal cytology who underwent colposcopy (±related procedures). METHODS: Questionnaires were mailed to women 4, 8 and 12 months post-colposcopy. Details of physical after-effects (pain, bleeding and discharge) experienced post-colposcopy were collected at 4 months. Colposcopy-specific distress was measured using the Process Outcome-Specific Measure at all time-points. Linear mixed-effects regression was used to identify associations between physical after-effects and distress over 12 months, adjusting for socio-demographic and clinical variables. MAIN OUTCOME MEASURES: Prevalence of post-colposcopy physical after-effects. Associations between the presence of any physical after-effects, awareness of after-effects, and number of after-effects and distress. RESULTS: Five-hundred and eighty-four women were recruited (response rate = 73, 59 and 52% at 4, 8 and 12 months, respectively). Eighty-two percent of women reported one or more physical after-effect(s). Multiple physical after-effects were common (two after-effects = 25%; three after-effects = 25%). Psychological distress scores declined significantly over time. In adjusted analyses, women who experienced all three physical after-effects had on average a 4.58 (95% CI: 1.10-8.05) higher distress scored than those who experienced no after-effects. Women who were unaware of the possibility of experiencing after-effects scored significantly higher for distress during follow-up. CONCLUSIONS: The prevalence of physical after-effects of colposcopy and related procedures is high. The novel findings of inter-relationships between awareness of the possibility of after-effects and experiencing multiple after-effects, and post-colposcopy distress may be relevant to the development of interventions to alleviate post-colposcopy distress. TWEETABLE ABSTRACT: Experiencing multiple physical after-effects of colposcopy is associated with psychological distress.

Adverse psychological outcomes following colposcopy and related procedures: a systematic review.

BACKGROUND: Although colposcopy is the leading follow-up option for women with abnormal cervical cytology, little is known about its psychological consequences. OBJECTIVES: We performed a systematic review to examine: (1) what, if any, are the adverse psychological outcomes following colposcopy and related procedures; (2) what are the predictors of adverse psychological outcomes post-colposcopy; and (3) what happens to these outcomes over time. SEARCH STRATEGY: Five electronic databases (PubMed, PsychINFO, CINAHL, Web of Science, Scopus) were searched for studies published in English between January 1986 and February 2014. SELECTION CRITERIA: Eligible studies assessed psychological wellbeing at one or more time-points post-colposcopy. DATA COLLECTION AND ANALYSIS: Two reviewers independently screened titles and abstracts. Full texts of potentially eligible papers were reviewed. Data were abstracted from, and a quality appraisal performed of, eligible papers. MAIN RESULTS: Twenty-three papers reporting 16 studies were eligible. Colposcopy and related procedures can lead to adverse psychological outcomes, particularly anxiety. Ten studies investigated predictors of adverse psychological outcomes; management type and treatment had no impact on this. Seven studies investigated temporal trends in psychological outcomes post-colposcopy; findings were mixed, especially in relation to anxiety and distress. Studies were methodologically heterogeneous. CONCLUSIONS: Follow-up investigations and procedures for abnormal cervical cytology can cause adverse psychological outcomes among women. However, little is known about the predictors of these outcomes or how long they persist. There is a need for a more standardised approach to the examination of the psychological impact of colposcopy, especially longer-term outcomes. TWEETABLE ABSTRACT: Follow-up investigations for abnormal cervical cytology can cause adverse psychological outcome among women.

Triage of LSIL/ASC-US with p16/Ki-67 dual staining and human papillomavirus testing: a 2-year prospective study.

OBJECTIVE: To investigate human papillomavirus (HPV) DNA testing and p16/Ki-67 staining for detecting cervical intraepithelial grade 2 or worse (CIN2+) and CIN3 in women referred to colposcopy with minor abnormal cervical cytology low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of undermined significance (ASC-US). The clinical performance of both tests was evaluated as stand-alone tests and combined, for detection CIN2+ and CIN3 over 2 years. METHODS: ThinPrep(®) liquid-based cytology (LBC) specimens were collected from 1349 women with repeat LSIL or ASC-US. HPV DNA was performed using Hybrid Capture. Where adequate material remained (n = 471), p16/Ki-67 overexpression was assessed. Clinical performance for detection of histologically diagnosed CIN2+ and CIN3 was calculated. RESULTS: Approximately 62.2% of the population were positive for HPV DNA, and 30.4% were positive for p16/Ki-67. p16/Ki-67 showed no significant difference in positivity between LSIL and ASC-US referrals (34.3% versus 28.6%; P = 0.189). Women under 30 years had a higher rate of p16/Ki-67 compared to those over 30 years (36.0% versus 26.6%; P = 0.029). Overall HPV DNA testing produced a high sensitivity for detection of CIN3 of 95.8% compared to 79.2% for p16/Ki-67. In contrast, p16/Ki-67 expression offered a higher specificity, 75.2% versus 40.4% for detection of CIN3. Combining p16/Ki-67 with HPV DNA improved the accuracy in distinguishing between CIN3 and

'I don't care whether it's HPV or ABC, I just want to know if I have cancer.' Factors influencing women's emotional responses to undergoing human papillomavirus testing in routine management in cervical screening: a qualitative study.

OBJECTIVE: To explore emotional responses, and predictors of negative reactions, among women undergoing human papillomavirus (HPV) tests in routine clinical practice. DESIGN: Exploratory qualitative interview study. SETTING: A large busy colposcopy clinic in a Dublin hospital. SAMPLE: Twenty-seven women who had had an HPV DNA test in the previous 6 months following one or more low-grade cytology tests or treatment for cervical intraepithelial neoplasia (CIN). METHODS: In-depth semi-structured interviews were conducted. Interview transcripts were analysed using a thematic approach (Framework Analysis). MAIN OUTCOME MEASURES: Women's emotional responses and predictors of negative emotional reactions. RESULTS: For most women, having a test for high-risk HPV types generated little negative or positive emotional impact. Adverse emotional responses related to HPV infection rather than testing. Factors that influenced whether women experienced negative emotional responses were: concerns over abnormal cytology or diagnosis of CIN; HPV knowledge; awareness of HPV being sexually transmitted; awareness of HPV prevalence; and HPV information needs. Women's concerns about abnormal cytology/CIN dominated all other issues. CONCLUSIONS: These qualitative data suggest that in the context of follow up of abnormal cytology or treatment for CIN, the emotional impact of HPV testing may be modest: women's primary concerns at this time relate to abnormal cytology/CIN.

CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity.

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells.

Kaposi's sarcoma (KS), a vascular tumour that contains characteristic spindle cells forming slit-like spaces, may have an infectious aetiology. Recently, sequences of a new human herpesvirus, KSHV/HHV-8, have been identified in both HIV-associated and classical KS. We sought to identify the target cell of this virus in KS tumour tissue. Using PCR in situ hybridization (PCR-ISH) we show that KSHV/HHV-8 is present in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical KS spindle cells. These findings show that KSHV/HHV-8 is present in the cell types thought to represent neoplastic cells in these lesions.

Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes.

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.

Early synthesis of specific cytoplasm proteins is correlated with the rate of exit of lymphocytes from the resting state.

We investigated the initiation of synthesis of proteins in human lymphocytes exposed to the mitogen phytohemagglutinin (PHA) for 6 h. Radiolabeled proteins in three subcellular fractions, cytoplasmic, nuclear salt wash, and nuclear, were separated on polyacrylamide gels. Compared with cells incubated for the same time in the absence of PHA only two cytoplasmic proteins of Mr 51 and 60 kd showed increased synthesis in a dose-dependent fashion. Synthesis of the 60-kd protein shows the strongest correlation with rate of entry into the first S phase and with rate of cellular aggregation. Thus, the 60-kd protein appears to be a major early response-associated protein for entry of lymphocytes into the first S phase after PHA stimulation.

RET/PTC rearrangement occurring in primary peritoneal carcinoma.

RET/PTC rearrangements are initiating events in the development of a significant proportion of papillary thyroid carcinomas. Activated RET/PTC mutations are thought to be restricted to thyroid disease, but this study proposes that these events may also occur in nonthyroid tumors. A total of 57 nonthyroid papillary tumors were examined for RET/PTC rearrangements using interphase fluorescence in situ hybridization, Taqman reverse transcriptase polymerase chain reaction, and immunohistochemistry. Taqman single nucleotide polymorphism detection was used to analyze for expression of mutated BRAF T1799A. In all, 20% (3/15) of primary peritoneal carcinoma had detectable RET/PTC1 rearrangements by all 3 methodologies. A further case of similar histotype had an alternate RET/ PTC rearrangement. No RET/PTC1 rearrangements were detected in the remaining tumor cohort. All 57 tumors were homozygous for wild-type BRAF. The results indicate that RET/PTC rearrangements occur in a small subset of nonthyroid papillary tumors. These rearrangements may not be directly implicated in tumor growth; rather representing "passenger" mutations reflecting RET instability in secondary tumor subclones.

Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy.

The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan(R) reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.

Squamous cell carcinoma of the head and neck in young Irish adults.

Classically, squamous cell carcinoma (SCC) of the head and neck is a disease of older adults, but recently there have been reports of an increasing incidence in young people. This study of patients in the Republic of Ireland compares sex distribution, sites, risk factors, stage and grade of tumour, and nodal status of 130 patients with SCC of the head and neck, 30 of whom were less than 40 years old. There was a highly significant association between age, smoking status, and site of tumour. For the first time to our knowledge in a study such as this, the preoperative haematological status of the patients was assessed, and although 15% were anaemic there was no significant difference in the occurrence of anaemia between the younger and the older patients. We think that it is possible that the biology of SCC of the head and neck in young people differs from that in older people.

PO-14 - Tumour expression of coagulation proteases of the aPC pathway - a role in the pathogenesis of gynaecological cancers?

INTRODUCTION: The close relationship between coagulation, thrombosis and cancer has long been established. Gynaecological cancers, in particular ovarian cancers, carry a high risk of thrombosis but coagulation activation is also thought to play a role in tumorigenesis and metastasis. In experimental animal models of metastasis, mice with a genetic procoagulant phenotype are prone to develop metastasis and anticoagulant therapy dramatically reduces pulmonary metastasis in these models. The aPC pathway is a key natural anticoagulant pathway, in addition to its role in venous thrombosis, dysregulation of this pathway is also thought to play a role in the pathogenesis of some cancers. No data exists in ovarian and endometrial cancers. AIM: The aim of this study is to determine the expression of key proteins of the activated protein C pathway in endometrial and ovarian malignant tumours compared to benign tumours and to assess their role in patient survival. MATERIALS AND METHODS: RNA was extracted from 78 (54 malignant and 24 benign) fresh frozen ovarian and endometrial tumours samples. Tumour biopsies were mRNA expression of endothelial protein C receptor (EPCR), protein S (PS), protein C (PC), thrombomodulin (TM), Factor V (FV) and VIII (FVIII) and PAR-1 and PAR-2 was measured using TaqMan Low Density Arrays. mRNA fold change relative to benign expression was determined using the 2 -delta delta Ct method with 18s as internal standard. All patients gave full and informed consent and the study had the approval of the hospital ethics committee. Total cell protein was extracted from ovarian tumour tissue. Enzyme-linked immunosorbent assay (ELISA) was used to measure protein plasma expression RESULTS: EPCR (P<0.001), protein S (P<0.0001) and Factor VIII (P<0.003) mRNA expression was significantly downregulated in malignant tumours compared with benign. Factor V and PAR-2 were significantly upregulated (P<0.001; P<0.004). Protein C was not consistently expressed. Reduced EPCR and TM protein expression was also observed in malignant tumours with increased plasma levels of Factor V. Reduced protein S and increased FV were associated with decreased survival. Plasma levels of Factor V were related to grade in the endometrial cancer group. PAR-2 mRNA expression was increased in ovarian tumours (P<0.001) however PAR-1 expression remained unchanged. CONCLUSIONS: Our results show reduced expression of key proteins associated with activation of protein C combined with increased expression in FV in gynaecological malignancies. These changes may contribute to local thrombin production and tumour progression and metastasis. Further work is required to determine the precise mechanisms involved.

Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins.

This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.

p16INK4A, CDC6, and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer.

AIM: To analyse and compare expression patterns of three potential biomarkers-p16(INK4A), CDC6, and MCM5-and evaluate their use as predictive biomarkers in squamous and glandular cervical preinvasive neoplasia. METHODS: Immunocytochemical analysis of p16(INK4A), MCM5, and CDC6 expression was performed on 20 normal, 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, 10 squamous cell carcinoma, 19 cervical glandular intraepithelial neoplasia (cGIN), and 10 adenocarcinoma samples. Staining intensity was assessed using a 0-3 scoring system. p16(INK4A), MCM5, and CDC6 expression was also examined in ThinPrep slides exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) was detected using a modified SYBR green assay. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. RESULTS: All three markers showed a linear correlation between expression and grade of dysplasia. p16(INK4A) and MCM5 protein expression was upregulated in all grades of squamous and glandular dysplasia. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinoma. CONCLUSION: p16(INK4A) expression was closely associated with high risk HPV infection-all grades of squamous and glandular cervical lesions were immunohistochemically positive. MCM5 staining intensity was independent of high risk HPV infection, highlighting its potential as a biomarker in both HPV dependent and independent cervical dysplasia. CDC6 may be a biomarker of high grade and invasive lesions of the cervix, with limited use in low grade dysplasia. p16(INK4A) was the most reliable marker of cervical dysplasia. Combinations of dysplastic biomarkers may be useful in difficult diagnostic cases.

Antiangiogenic effects of camptothecin analogues 9-amino-20(S)-camptothecin, topotecan, and CPT-11 studied in the mouse cornea model.

Angiogenesis has been correlated with increased invasion and metastases in a variety of human neoplasms. Inadequate inhibition of the growth of tumor microvessels by anticancer agents may result in treatment failure, rated clinically as progressive or stable disease. We have investigated the antiangiogenic properties of three camptothecin analogues, 9-amino-20(S)-camptothecin, topotecan, and camptosar (CPT-11), currently under investigation in clinical settings. Angiogenesis was induced by basic fibroblast growth factor in the cornea of inbred Swiss-Webster mice, with the aim of exploring the suppression of neovascularization by the analogues injected into the mice daily over a period of 6 days. The dose range chosen is known to inhibit, in the mouse model, the growth of various human tumor xenografts or murine tumors. The statistical analysis evaluated the association between the area of neoangiogenesis and the dose of the drugs tested and correlated the effects with observed drug toxicity. It was established that, as the drug doses increased, the area of neovascularization decreased, appearing to approximate a negative exponential curve. 9-Amino-20(S)-camptothecin at 6.89 and 8.26 micromol/kg (2.5 and 3.0 mg/kg) and topotecan at 8.31 micromol/kg (3.5 mg/kg), both drugs being delivered over a 6-day period, had statistically significant reduction (47.2-72.5%) of neoangiogenesis and acceptable toxicity. At higher doses of the two analogues, toxic body-weight losses and deaths were observed. CPT-11 showed statistically significant reduction of neoangiogenesis at a dose of 359 micromol/kg (210 mg/kg) delivered over a 6-day course. Unlike camptothecin analogues, the nontoxic dose of vincristine did not induce a statistically significant inhibition of angiogenesis, and there was no dose-dependent escalation of antiangiogenic effects. The results indicate that camptothecins are most likely cytotoxic against two tumor compartments: in addition to tumor cells of epithelial origin, the drugs act against endothelial cells and prevent the growth of the tumor microvessels. We have hypothesized that treatment failure in some patients is due to incomplete or inadequate inhibition of the microvessel growth by camptothecins. Presumably, an intensive inhibition of the remaining tumor microvasculature in such patients could be achieved by combining a camptothecin with another antiangiogenic anticancer agent or with a highly selective angiogenic inhibitor exerting minimal dose-limiting toxicity. Such treatment by a camptothecin plus a less toxic inhibitor of angiogenesis can improve antitumor efficacy. To validate this concept, preclinical studies followed by clinical trials are planned.

ret/PTC and BRAF act as distinct molecular, time-dependant triggers in a sporadic Irish cohort of papillary thyroid carcinoma.

The purpose of this study was to assess BRAF mutation rates in various thyroid tissues and to investigate if concomitant mutations with ret/PTC activation occurred in inflammatory and neoplastic lesions. To this end, we developed a novel Taqman based screening assay for the common T1799A BRAF mutation. Heterozygous T1799A mutations were detected in 13 of 34 (44%) papillary thyroid carcinomas (PTCs) tested. No such mutations were detected in the other tissue types tested. Concomitant presence of both oncogenes was reported in 5 of the 34 PTCs. A significant temporal trend was observed, with ret/PTC chimera detected for the most part before 1997 and BRAF mutations being more prevalent after 1997. The results suggest that some environmental/etiological agent(s) may have influenced the pathobiology of thyroid tumor development, among the population examined, over time.

Evidence for expansion of a population of lymphocytes with reduced or absent T3 expression in aged human donors.

In a previous report, we described an unusual pattern of T cell associated surface marker expression in unfractionated mononuclear cells from aged donors; an excess of T4 and T8 positive cells relative to T3 positive cells. This study further characterizes these cells on the basis of density, adherence to nylon wool and quantitative expression of cell surface markers. We find that the population of lymphocytes responsible for the unusual surface marker expression is of low density, adheres to nylon wool, and is present in small numbers in young donors. The adherent cells have a reduced quantitative expression of the T3 antigen, no change in the antigen density of T4 and T8, and have increased expression of the T10 antigen. These cells do not have the characteristics of large granular lymphocytes, monocytes, B cells with unusual marker expression, or thymocytes poised for export to peripheral blood. We suggest that these cells, found in increased numbers in aged humans, may represent an expansion of a population of T lymphocytes with absent or reduced T3 antigen expression found normally in smaller numbers in young adults. T lymphocyte antigen receptor density has been quantitatively linked to expression of the T3 antigen. Thus, our results imply that aging may lead to decreased T cell surface antigen density, which may account in part for decline in T cell function with age.

Expression of the human T cell antigen receptor complex in advanced age.

Several studies of T cell-associated surface marker expression in older subjects "with no evidence of diseases known to affect immune function" have suggested that diminished expression of the constant portion (CD3) of the human T cell antigen-receptor complex (CD3/Ti) may be associated with aging. To determine if there is a decrease with age in expression of this complex, we determined the percent of cells positive for WT31, an antibody that recognizes a constant epitope on Ti alpha-beta heterodimers, CD3, CD4 and CD8 by immunofluorescence on peripheral blood lymphocytes from young controls (19-31 years), and 2 well characterized populations of older donors (greater than 69 years). The first group of older donors had no history of chronic or recent acute illness and saw a physician only for routine medical care. The second group was selected from patients seen in a geriatrics clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. There were no significant differences in the percent of cells positive with WT31, or with antibodies to CD4 and CD8 between young adults and the 2 groups of older donors. The clinic subjects showed a consistent decrease in percent of CD3+ lymphocytes in peripheral blood mononuclear cells compared to young adults (5-10% reduction) with less difference between the healthy older subjects and young adults. In the clinic subjects this reduction appears to be due to a decrease in CD3+, WT31-lymphocytes. These cells represent about 5-6% of the total T cell population in the young and healthy older group and are known to express CD3 in combination with Ti gamma-delta dimers. We show here that these cells are about equally distributed in high and low density lymphocytes after Percoll fractionation. We conclude that significant quantitative reduction in expression of the CD3/Ti antigen-receptor complex on human T cells is not a feature of the primary aging process and cannot account for diminished in vitro proliferative response of healthy subjects with age. However, diminished abundance of T cells expressing products of the Ti gamma-chain in less healthy older subjects may predispose to or result from diseases not usually associated with altered immune function.